In a healthy mammal, the pancreas produces and secretes enzymes that digest carbohydrates, fats and proteins in the gastrointestinal tract. They are produced in inactive form (termed proenzyme) and subsequently are converted to an active form in the small intestine giving rise to a cascade of amylolytic, lipolytic and proteolytic activity. The cascade is thought to begin with the conversion of pancreatic trypsinogen to trypsin catalyzed by enterokinase, a proteolytic enzyme associated with the small intestine. The newly-formed trypsin then converts the other pancreatic proenzymes into their active forms to trigger a burst of enzymatic activity characterized as digestion. Greenberger et al., Diseases of the Pancreas, "Harrison's Principles of Internal Medicine," 11th Edition, p. 1372, McGraw-Hill, (E. Brunewald et al. edit, 1987).
In the absence of perturbing factors, the pancreas is able to protect itself from the autodigestion which could result from the digestive enzymes it produces. The pancreatic acinar cells (where the digestive enzymes are synthesized and stored) provide three control mechanisms to prevent their own destruction. First, the enzymes are produced as catalytically inactive proenzymes. Second, after their synthesis, but before secretion into the digestive system, the enzymes are segregated from the acinar cell cytoplasm in lysosomes (membrane-bound intracellular organalles). Third, the lysosomes containing the enzymes contain potent protein inhibitors of trypsin which prevent premature activation of other hydrolyric proenzymes. Steer el al., New England Journal of Medicine, 316:144 (1987).
In the presence of perturbing factors, a disorder of the pancreas termed pancreatitis (either acute or chronic) may result. Acute pancreatitis can manifest itself as a mild and self-limiting disorder (termed edematous pancreatitis), or in a more severe form (termed necrotizing pancreatitis) where permanent damage to pancreatic tissue occurs. Chronic pancreatitis results in extensive and permanent destruction of the pancreas. Greenberger et al., supra, at 1372.
Acute pancreatitis is usually associated with biliary tract stones, while chronic pancreatitis is often associated with chronic alcohol abuse. Steer et al., supra, at 144. Pancreatitis may also arise as a complication of cardiac surgery involving cardiopulmonary bypass procedures, and is reported to follow all types of open-heart surgery, including cardiac transplantation. Castillo et al., New England Journal of Medicine, 325:382 (1991). Moreover, bouts of acute pancreatitis are occasionally induced following gastrointestinal diagnostic and surgical procedures, such as bile duct exploration, sphincteroplasty, distal gastrectomy, splenctomy, and endoscopic retrograde cholangiopancreatography. Bardenheier et al., Am. J. Surg. 116:773 (1968); Cotton, Gut, 18:316 (1977). Significant pancreatic injury has been reported in 1 to 3% of patients suffering from abdominal trauma which occasionally results in obstructive chronic pancreatitis.
Pancreatitis is characterized by damage to the pancreas and surrounding tissues which arises from autodigestion of the cells by the various digestive enzymes activated by trypsin. Animal studies of chemically-induced pancreatitis suggest that the disorder is rooted in the inability of pancreatic acinar cells to excrete the digestive proenzymes. This results in the activation of trypsinogen to trypsin by lyosomal hydrolases within the cell, with the amount produced exceeding protective levels of protease inhibitor normally available. Steer et al., supra, at 148; Gabryelewicz, Digestion, 40:19 (1988). This results in the subsequent activation of the other digestive enzymes co-localized with trypsin in the lysosome. These activated digestive enzymes cause edema, interstitial hemorrhage, vascular damage, coagulation necrosis, fat necrosis and parenchymal cell necrosis, Greenberger et al., supra, at 1372.
The activated digestive enzymes may subsequently enter the blood and the peritoneal cavity and can lead to secondary multiple organ damage. Although the blood contains trypsin inhibitors, it has been reported that trypsin complexed with one such inhibitor, alpha-2-macroglobulin, remains active. Rinderknecht et al., Biochim. Biophys. Acta, 295:233 (1973); Harpel et al., J. Clin. Invest., 52:2175 (1975); Rinderknect et al., Biochim. Biophys. Acta, 377:158 (1975). This active complex is thought to contribute in part to the metastatic proteolytic damage observed in pancreatitis. Jones, et al., Gut, 23:939 (1982).
A number of compounds have been examined for treatment of pancreatitis. Specifically, aprotinin, Futhan, Foy, Foy-305 and the leupeptins.
Aprotinin is a polypeptide of 58 amino acids and is reported to be a potent inhibitor of trypsin, with a dissociation constant (K.sub.d) of 3.times.10.sup.-11 M. Jones, supra, at p. 939. However, it is also reported to be ineffective in the treatment of human acute pancreatitis. Imrie et al., Br. J. Surg., 65:337 (1978); M.C.R. Working Party, Lancet, ii: 632 (1977); Niederau et al., J. Clin. Invest., 78:1056, 1061 (1966). Aprotinin is also an inhibitor of the coagulation factors, kallikrein and plasmin with a K.sub.d of 1.times.10.sup.-7 M and 2.times.10.sup.-10 M respectively. Kassell et al., Biochem. Biophys. Res. Commun., 18:225 (1965); Fritz el al., Arzneim. Forsch. 33:479 (1983); Lazdunski et al., Proteinase Inhibitors, p 420, Springer Verlag (H. Fritz et al. ed. 1974); Trautschold et al., Biochem. Pharmacol., 16:59 (1967).
Futhan is a nonpeptidyl low molecular weight protease inhibitor first synthesized by Fuji et al., Biochim. Biophys. Acta, 661:342 (1981). It is also known as nafamstat mesilate, FUT-175, and 6-amidino-2-naphthyl-4-guanidino benzoate dimethanesulfonate. It is reported to be effective in the treatment of acute pancreatitis induced in animal models. Iwaki et al., Jap. J. Pharmac. 41:155 (1986); Gabryelwicz et al., supra, at p 22. It is a potent inhibitor of trypsin, as well as the coagulation enzymes, kallikrein, factor Xa, and thrombin. Aoyama et al., Japan J. Pharmacol., 35:203 at 209 (1984); Hitomi et al., Hemostasis, 15:164 (1985).
Foy (also known as gabexate mesilate) and Foy-305 (also known as camostate) are also nonpeptidyl low molecular weight protease inhibitors. Both are reported to be effective to varying degrees in the treatment of acute pancreatitis induced in animal models. Niederau, supra, at 1061; Lankisch et al., Gastroenterology, 96:193 (1989). Both compounds are reported to be effective inhibitors of trypsin, as well as the coagulation/fibrinolysis enzymes, kallikrein, thrombin, plasmin and Clr complement system enzyme. Muramutu et al., Biochim. Biophys. Acta, 268:221 (1972); Takasugi et al., J. Med. Sci., 29:188 (1980); Tamura et al., Biochim. Biophys. Acta, 484:417 (1977).
The leupeptins are low molecular weight peptidyl aldehydes consisting of N-acetyl-L-leucyl-L-leucyl-L-argininal, N-propionyl-L-leucyl-L-leucyl-L-argininal and their analogs which contain L-isoleucine or L-valine in place of L-leucine. Aoyagi et al., "Structures and Activities of Protease Inhibitors of Microbial Origin", Proteases and Biological Control, pp. 429-454, Cold Spring Harbor Laboratory. Press (Reich et al. edit. 1975). They are reported to prolong the survival of rats in which acute pancreatitis has been induced. Jones, supra, at p. 939. They are potent inhibitors of trypsin and other serine proteases. Chiet al., J. Antibiotics, XLII: 1506 (1989).
Various derivatives of the leupeptins have been disclosed which are also potent inhibitors of trypsin. N-benzyloxycarbonyl-L-pyroglutamyl-L-leucyl-L-argininal was shown to be a potent inhibitor of trypsin (with an IC.sub.50 about 7 times lower than than that for N-acetyl-L-leucyl-L-leucy-L-argininal) (Saino et al., J. Antibiotics, XLi: 220 (1988)).
U.K. Patent Application 2,153,825 and Japanese Application 60-163815 describe naphthalene derivatives of arginine as trypsin inhibitors useful for treatment of pancreatitis; and Niederau et al., Gastroenterology 88:1192 (1985) describe proglumide, benzotript and secretin as protective agents against caerulein-induced pancreatitis in mice.